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Gene expression under normoxia/hypoxia in mouse embryonic fibroblasts with mutations in the CH1 domains of p300 and CBP

ABSTRACT: The CH1 (TAZ) domain of the transcriptional coactivators p300 and CBP has been reported to interact with the transcription factor HIF-1alpha and this interaction is thought to be critical for HIF-1alpha target gene expression in response to hypoxia. To determine the requirement for the CH1 domain in hypoxia-responsive gene expression, primary mouse embryonic fibroblasts (MEFs) were generated from e14.5 C57B/6x129/Sv F2 embryos that were either wildtype or bore deletion mutations in the CH1 protein binding domains of both alleles of p300 and one allele of CBP (tri_CH1). Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction. Keywords: genetic modification, dose response

ORGANISM(S): Mus musculus

PROVIDER: GSE3195 | GEO | 2005/10/24

SECONDARY ACCESSION(S): PRJNA92855

REPOSITORIES: GEO

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Transcription profiling of mouse embryonic fibroblasts C57Bl/6x129/Sv F2 e14.5 embryos vs controls grown in hypoxic or normoxic conditions

Project description:The CH1 protein interaction domain of the transcriptional coactivators p300 and CBP is thought to interact with HIF-1alpha and this interaction is thought to be critical to the expression of HIF-1alpha target genes in response to hypoxia. To test the requirement of the CH1 domain for gene expression in response to hypoxia, rimary mouse embryonic fibroblasts (MEFs) were generated from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300. The remaining allele of p300 or CBP was a conditional knock out allele. Control MEFs with only a single conditional knockout allele of p300 or CBP were also generated. At passage 3 MEFs were infected with Cre Adenovirus and grown until they had expanded at least 100 fold. Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction. 12 samples; 4 genotypes [CBP+/flox (flox1), p300 +/flox (flox2), CBP CH1/flox;p300 CH1/CH1 (triCH1flox1),CBP CH1/CH1;p300 CH1/flox (triCH1flox2)] , 2 treatments (normoxia and hypoxia).

2007-12-15 | E-GEOD-3196 | biostudies-arrayexpress

Transcription profiling of mouse embryonic fibroblasts normoxia/hypoxia treatments with mutations in the CH1 domains of p300 and CBP

Project description:The CH1 (TAZ) domain of the transcriptional coactivators p300 and CBP has been reported to interact with the transcription factor HIF-1alpha and this interaction is thought to be critical for HIF-1alpha target gene expression in response to hypoxia. To determine the requirement for the CH1 domain in hypoxia-responsive gene expression, primary mouse embryonic fibroblasts (MEFs) were generated from e14.5 C57B/6x129/Sv F2 embryos that were either wildtype or bore deletion mutations in the CH1 protein binding domains of both alleles of p300 and one allele of CBP (tri_CH1). Subconfluent MEFs were treated with 21% oxygen (normoxia) or 0.1% oxygen (hypoxia) with 5% carbon dioxide at 37 C in a humid chamber for 6hrs. At the start of treatment, medium was removed and replaced with medium (DMEM+10% FBS+pen-strep+ l-glu) that had been preequilibrated overnight in normoxia or hypoxia as appropriate. Immediately after treatment, cells were lysed in Trizol for RNA extraction. Experiment Overall Design: 4 samples: two genotypes (1 control, 1 mutant); two treatments (normoxia, hypoxia).

2008-06-12 | E-GEOD-3195 | biostudies-arrayexpress

Gene expression in hypoxic MEFs having only p300 and CBP with deleted CH1 domains

2005-10-24 | GSE3196 | GEO

Transcription profiling of primary mouse embryonic fibroblasts (MEFs) from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300

Project description:The CH1 protein interaction domain of the transcriptional coactivators p300 and CBP is thought to interact with HIF-1alpha and this interaction is thought to be critical to the expression of HIF-1alpha target genes in response to hypoxia. Trichostatin A (TSA), an inhibitor of histone deacetylases, has been reported to repress the expression of HIF-1alpha target genes. To test the requirement of the CH1 domain and TSA for gene expression in response to dipyridyl (a hypoxia mimetic), primary mouse embryonic fibroblasts (MEFs) were generated from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300. The remaining allele of p300 or CBP was a conditional knock out allele. Control MEFs with only a single conditional knockout allele of p300 or CBP were also generated. At passage 3 MEFs were infected with Cre Adenovirus and grown until they had expanded at least 100 fold. Subconfluent MEFs were treated with ethanol vehicle or 100ng/ml TSA with 5% carbon dioxide at 37 C in a humid chamber for 30 min., followed by ethanol vehicle or 100 umdipyridyl (DP) for an additional 3hrs. Immediately after treatment, cells were lysed in Trizol for RNA extraction.

2007-07-07 | E-GEOD-3296 | biostudies-arrayexpress

Gene expression in MEFs in response to treatment with dipyridyl and trichostatin A

Project description:The CH1 protein interaction domain of the transcriptional coactivators p300 and CBP is thought to interact with HIF-1alpha and this interaction is thought to be critical to the expression of HIF-1alpha target genes in response to hypoxia. Trichostatin A (TSA), an inhibitor of histone deacetylases, has been reported to repress the expression of HIF-1alpha target genes. To test the requirement of the CH1 domain and TSA for gene expression in response to dipyridyl (a hypoxia mimetic), primary mouse embryonic fibroblasts (MEFs) were generated from C57Bl/6x129/Sv F2 e14.5 embryos that contain a deletion in the CH1 domain of three of four alleles of CBP and p300. The remaining allele of p300 or CBP was a conditional knock out allele. Control MEFs with only a single conditional knockout allele of p300 or CBP were also generated. At passage 3 MEFs were infected with Cre Adenovirus and grown until they had expanded at least 100 fold. Subconfluent MEFs were treated with ethanol vehicle or 100ng/ml TSA with 5% carbon dioxide at 37 C in a humid chamber for 30 min., followed by ethanol vehicle or 100 umdipyridyl (DP) for an additional 3hrs. Immediately after treatment, cells were lysed in Trizol for RNA extraction. Keywords: genetic modification, dose response

2005-10-24 | GSE3296 | GEO

Transcription profiling of mouse embryonic fibroblasts from embryos with ?CH1 mutation in p300 and/or CBP atreated with hypoxia or dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) reveals two transactivation mechanisms are responsible for the bulk of HIF-1alpha-responsive gene expression

Project description:The C-terminal activation domain (C-TAD) of the hypoxia-inducible transcription factors HIF-1? and HIF-2? binds the CH1 domains of the related transcriptional coactivators CREB-binding protein (CBP) and p300, an oxygen-regulated interaction thought to be highly essential for hypoxia-responsive transcription. The role of the CH1 domain in vivo is unknown, however. We created mutant mice bearing deletions in the CH1 domains (?CH1) of CBP and p300 that abrogate their interactions with the C-TAD, revealing that the CH1 domains of CBP and p300 are genetically non-redundant and indispensable for C-TAD transactivation function. Surprisingly, the CH1 domain was only required for an average of ~35-50% of global HIF-1?-responsive gene expression, whereas another HIF-transactivation mechanism that is sensitive to the histone deacetylase inhibitor trichostatin A (TSAS) accounts for ~70%. Both pathways are required for greater than 90% of the response for some target genes. Our findings suggest that a novel functional interaction between the protein acetylases CBP and p300, and deacetylases, is essential for nearly all HIF-responsive transcription. Experiment Overall Design: Three separate affymetrix experiments using mouse embryonic fibroblasts derived from embryos bearing the ?CH1 mutation in p300 and/or CBP and treated with hypoxia or combinations of dipyridyl (a hypoxia mimetic) and trichostatin A (a histone deacetylase inhibitor) are described (GSE3195, GSE3196 and GSE3296). Samples are not directly comparable between experiments because of differences in experiment design and Affymetrix chips used.

2008-06-12 | E-GEOD-3318 | biostudies-arrayexpress

Two transactivation mechanisms are responsible for the bulk of HIF-1alpha-responsive gene expression

2005-10-24 | GSE3318 | GEO

MYCN directly regulates TET1 expression to control 5-hmC distribution and enhance neuroblastoma cell migration in hypoxia [ChIP-Seq]

Project description:We performed HIF-1alpha ChIP-seq in SK-N-BE(2) cells cultured in normoxia vs. hypoxia for 48 hrs. We identified two new HIF-1alpha binding sites in TET1 that control TET1expression in hypoxia

2022-08-10 | GSE167477 | GEO

HIF-1 directly induces TET3 expression to redistribute 5-hydroxymethylcytosine and induce erythroid gene expression in hypoxia [ChIP-seq]

Project description:We performed HIF-1alpha ChIP-seq in K562 cells cultured in normoxia vs. hypoxia for 3 days. We identified two new HIF binding sites in TET3 intron 2 that control TET3 expression in hypoxia

2020-02-01 | GSE142865 | GEO

Transcription profiling of C. elegans hypoxia responses

Project description:The goal of the study was to identify hypoxia-induced gene expression changes in C. elegans and to determine which of these responses to hypoxia were regulated by hif-1. Towards these aims, we analyzed mRNA expression patterns in synchronized populations of wild-type worms that were cultured in standard lab conditions (Snormoxia) or in hypoxia. We also assayed mRNA from two mutant strains: (i) animals carrying the strong loss-of-function mutation in hif-1 and (ii) C. elegans that carry a deletion in vhl-1 and express the HIF-1 protein at constitutively high levels. In the microarray experiments, 3 strains were assayed: wild type N2, hif-1 (ia04), and vhl-1 (ok161). Worms were incubated for 4 hours in 21% oxygen or 0.1% oxygen at 210C. Animals were quickly harvested in ice cold M9 buffer, and poly (A) RNA was isolated using established procedures. No more than 3 minutes elapsed between the removal of plates from the hypoxic chamber and the addition of Trizol. RNA was isolated from three independent experiments for each experimental condition (3 genotypes; normoxia vs hypoxia). mRNAs were hybridized to whole-genome microarrays which contained probes for 17,817 predicted genes (94% of the genome). Ch1 in the array reprents the data for the worms growed under hypoxia; and Ch2 in the array reprents the data for the worms growed under normoxia.

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